The appropriate cell seeding density for live/dead cell imaging depends on various factors such as cell type, size of the wells, and desired confluency level. However, a general guideline is to seed cells at a density that allows them to reach confluence within 24-48 hours. This ensures that the cells are in log phase growth during the imaging period, which is important for accurate assessment of cell proliferation.

For a 24-well plate, you can seed cells at a density of around 10^5 - 10^6 cells per mL. This will allow the cells to grow and reach confluence within 24-48 hours, depending on the cell type and growth conditions.

To calculate the number of cells needed for each well, consider the following formula:

Number of cells = Volume of media (in mL) x Cell concentration (cells/mL)

For example, if you plan to use 1 mL of media per well and want to seed 10^5 cells/mL, then you would need:

Number of cells = 1 mL x 10^5 cells/mL = 10^5 cells

Therefore, you would need to add 10^5 cells to each well of the 24-well plate.

It's worth noting that this is just an estimate, and the actual cell seeding density may vary based on your specific experimental design and cell type. It's always a good idea to perform a pilot experiment to determine the optimal cell seeding density for your particular assay.

There is no single ideal cell seeding density for live/dead cell imaging to assess proliferation, as it depends on several factors:

- proliferation rates can vary widely between different cell types.

- The doubling time of the cells - this determines how quickly the cells will proliferate and reach confluence.

- Whether you want to capture exponential or linear growth phases.

A reasonable starting point would be to seed at a low density, such as 5,000-10,000 cells per cm2. For a 24-well plate, that equals ~10,000 - 20,000 cells per well. This should provide enough room for exponential growth for rapidly proliferating cell types over 72 hours without overcrowding.

However, it's best to first determine the doubling time for your cell type. Then do a pilot experiment with different seeding densities to determine the optimal range that avoids overconfluence at the endpoint while still yielding enough cells for imaging.

Hello Noor Ul Ain

It would be difficult to mention the seeding density for a 24 well plate because for each cell type the seeding density will vary depending on the doubling time, growth condition available and the heath status of the cells.

So, I would recommend that you perform a cell density titration assay to determine the optimum cell number for seeding such that the cells do not become 100% confluent at the end of 72 hours. This is necessary because if the cells become overconfluent at the end of 72 hours, interpretation of results would become difficult. So, aim for around 80-85% confluency at the end of 72 hours.

Also, carefully monitor the cell density or confluency of your cells prior to their use in the experiment as this will help prevent unintended cellular stress exposure that could affect your end results.

Best.

Thank you so much everyone for your pieces of advice.

Malcolm Nobre for scratch wound assay I used 24 well plate and cell seeding was 10^5 cells/well and it reached full confluency after 24 h of seeding. I am using human fibroblasts.

Now I want to do the live dead cell imaging, either 24, 48 or 96 well plate, which is highly recommended for live/dead cell imaging?

Noor Ul Ain,

24-well plate will do. But I would prefer 96-well plate because it will give one the ability to record the whole well area with just one image without the need for tilting or distortion. By using a 96-well plate you will not miss any cell while investigating the entire cell population in the microplate well. If you have enough experience in cell culture and confident to handle a microplate, you may try 96-well plate, otherwise 24-well plate would be much easier to work with.

Best.

Malcolm Nobre thank you so much for your advice. I am a beginner in cell culturing so mostly I rely on the literature and follow what they have used. That's why I ask when I am not confident about something.

No problem. Wish you all the best!

Malcolm Nobre thank you so much!

You're welcome.